Multi-omics integrated analyses indicated that non-polysaccharides of Sijunzi decoction ameliorated spleen deficiency syndrome via regulating microbiota-gut-metabolites axis and exerted synergistic compatibility.

ETHNOPHARMACOLOGICAL RELEVANCE: As a classical traditional Chinese medicine formula to invigorating spleen and replenishing qi, Sijunzi decoction (SJZD) is composed of four herbs, which is applied to cure spleen deficiency syndrome (SDS) clinically. The non-polysaccharides (NPSs) of SJZD (SJZD_NPS) are important pharmacodynamic material basis. However, the amelioration mechanism of SJZD_NPS on SDS has not been fully elaborated. Additionally, the contribution of herbs compatibility to efficacy of this formula remains unclear. AIM OF THE STUDY: The aim was to explore the underlying mechanisms of SJZD_NPS on improving SDS, and uncover the scientific connotation in SJZD compatibility. MATERIALS AND METHODS: A strategy integrating incomplete formulae (called "Chai-fang" in Chinese) comparison, pharmacodynamics, gut microbiome, and metabolome was employed to reveal the role of each herb to SJZD compatibility against SDS. Additionally, the underlying mechanism harbored by SJZD_NPS was further explored through targeted metabolomics, network pharmacology, molecular docking, pseudo-sterile model, and metagenomics. RESULTS: SJZD_NPS significantly alleviated diarrhea, disordered secretion of gastrointestinal hormones and neurotransmitters, damage of ileal morphology and intestinal barrier in SDS rats, which was superior to the NPSs of Chai-fang. 16S rRNA gene sequencing and metabolomics analyses revealed that SJZD_NPS effectively restored the disturbed gut microbiota community and abnormal metabolism caused by SDS, showing the most evident recovery. Moreover, SJZD_NPS recalled the levels of partial amino acids, short chain fatty acids and bile acids, which possessed strong binding affinity towards potential targets. The depletion of gut microbiota confirmed that the SDS-amelioration efficacy of SJZD_NPS is dependent on the intact gut microbiome, with the relative abundance of potential probiotics such as Lactobacillus_johnsonii and Lactobacillus_taiwanensis been enriched. CONCLUSION: NPSs in SJZD can improve SDS-induced gastrointestinal-nervous system dysfunction through regulating microbiota-gut-metabolites axis, with four herbs exerting synergistic effects, which indicated the compatibility rationality of SJZD.

The compatibility rationality of Sijunzi decoction based on integrated analysis of tissue distribution and excretion characteristics in spleen deficiency syndrome rats.

ETHNOPHARMACOLOGICAL RELEVANCE: As a classical prescription for treating spleen deficiency syndrome (SDS), Sijunzi decoction (SJZD) is composed of Ginseng Radix et Rhizoma (RG, Panax ginseng C.A.Mey.), Atractylodes Macrocephalae Rhizoma (AM, Atractylodes macrocephala Koidz.), Poria (Poria cocos (Schw.) Wolf) and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle (GRP, processed from Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L.). The non-polysaccharides (NPSs) are the pharmacodynamic substance basis of SJZD, whose pharmacokinetics in SDS rats were elaborated previously. Further study on their tissue distribution and excretion properties is of significance for understanding the compatibility laws of SJZD. AIM OF THE STUDY: The aim was to unravel the tissue distribution and excretion characteristics of NPSs of SJZD in SDS rats, and explore the scientific connotation of SJZD compatibility. MATERIALS AND METHODS: A validated ultrafast liquid chromatography tandem mass spectrometry method was developed for monitoring the accurate dynamics of sixteen components in the tissues, feces and urine of SDS rats. The four incomplete formulae of SJZD were prepared by randomly deleting one herb to uncover the herb-herb interactions. RESULTS: All components of NPSs in SJZD were distributed in the tissues, except for ononin in the heart. Among them, glycyrrhetinic acid and atractylenolide III were more abundant in the liver and lung, respectively, while other components were enriched in the ileum, especially saponins. The evaluation of fecal excretion and urinary excretion revealed the low cumulative excretion of all components. The comparative analysis of incomplete formulae indicated that the tissue distribution and excretion became faster after removing Poria from SJZD, while a lack of RG led to slower tissue distribution. The tissue distribution at most time points was reduced when AM was absent. Further comprehensive visualization implied that SJZD compatibility can improve tissue distribution of the NPSs, especially ginsenosides and atractylenolide, at the specific time periods. CONCLUSION: The tissue distribution and excretion characteristics of NPSs of SJZD were elucidated in current research. Meanwhile, this study proposed new insights into the mechanism of SJZD compatibility rationality.

Multiple approaches to characterize and visualize the chemical composition of Sijunzi Decoction comprehensively.

Sijunzi Decoction is composed of Ginseng Radix et Rhizoma, Atractylodes Macrocephalae Rhizoma, Poria, and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, and it is a classic formula for treating spleen deficiency syndrome in Chinese medicine. Clarifying the active substances is an effective way to develop Traditional Chinese medicine and innovative medicines. Carbohydrates, proteins, amino acids, saponins, flavonoids, phenolic acids, and inorganic elements in the decoction were analyzed by multiple approaches. A molecular network was also used for visualizing the ingredients in Sijunzi Decoction, and representative components were also quantified. The detected components accounted for 74.544% of the Sijunzi Decoction freeze-dried powder, including 41.751% crude polysaccharides, 17.826% sugars (degree of polymerization 1-2), 8.181% total saponins, 2.427% insoluble precipitates, 2.154% free amino acids, 1.177% total flavonoids, 0.546% total phenolic acids, and 0.483% inorganic elements. Molecular network and quantitative analysis used to characterize the chemical composition of Sijunzi Decoction. The present study systematically characterized the constituents of Sijunzi Decoction, revealed the composition ratio of each type of constituent, and provided a reference for study on the substance basis of other Chinese medicine.

Drug-polysaccharide/herb interactions and compatibility rationality of Sijunzi decoction based on comprehensive pharmacokinetic screening for multi-components in rats with spleen deficiency syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Sijunzi decoction (SJZD) is composed of four herbs, namely Ginseng Radix et Rhizoma (RG, Panax ginseng C.A.Mey.), Atractylodes Macrocephalae Rhizoma (AM, Atractylodes macrocephala Koidz.), Poria (Poria cocos (Schw.) Wolf), and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle (GRP, derived from Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L.) based on the compatibility theory of traditional Chinese medicine (TCM), which is a classical formula for the treatment of spleen deficiency syndrome (SDS) in TCM. The polysaccharides and non-polysaccharides (NPSs) composition represented by flavonoids, saponins and terpenoids are the important pharmacodynamic material basis of SJZD. AIM OF THE STUDY: The aim of this study was to investigate the pharmacokinetic characteristics of SJZD in normal rats and SDS rats, and explore the potential interactions between NPSs and polysaccharides in SJZD, as well as the compatibility rationality of SJZD. MATERIALS AND METHODS: SDS model was established by oral administration of Radix Rhei (Rheum officinale Baill.) extract, loaded swimming, and intermittent fasting. A rapid, sensitive and reliable ultrafast liquid chromatography tandem mass spectrometry (UFLC-MS/MS) method was developed for the simultaneous analysis of fifteen representative compounds in rat plasma to investigate the differences in pharmacokinetics between normal and SDS rats. The SJZD-NPS samples were prepared by removing the polysaccharides of SJZD to explore the interactions between NPSs and polysaccharides of SJZD. According to the compatibility theory of TCM, four incomplete formulae of SJZD were obtained by randomly removing an herb (also called 'que fang' in TCM), and their pharmacokinetic differences were compared to elucidate the rationality of SJZD compatibility with oral administration to SDS rats. RESULTS: The established UFLC-MS/MS method showed perfect performance in simultaneously analyzing fifteen compounds of SJZD in rat plasma. Compared with normal rats, the absorption efficiency of NPSs in SDS rats was lower, accompanied by the prolonged residence time (Cmax and AUC0-t reduced, while MRT0-t increased). Polysaccharides have the potential to enhance intestinal metabolism of glycosides among these components, thereby contributing to the circulating distribution of corresponding metabolites (e.g. aglycones). Furthermore, the compatibility of the four herbs in SJZD could alter their pharmacokinetic characteristics, and potentially improve the absorption of the effective components of RG and AM, which is in accordance with the principle that "monarch" and "minister" herbs play a major role in TCM. In detail, the improved absorption of ginsenosides was mainly regulated by GRP (the "guide" herb in SJZD), together with the effects of AM ("minister" herb) and Poria ("adjuvant" herb) on the pharmacokinetics of components in GRP, implying that herb-herb interactions existed in SJZD and demonstrated the compatibility rationality of SJZD potentially. CONCLUSION: This study laid a solid foundation for revealing the pharmacodynamic material basis and subsequent action mechanism of SJZD, as well as provided new insights into the compatibility of SJZD. The comprehensive pharmacokinetic approach adopted in the current research also provides a valuable strategy for TCM formulae research.

Pharmacodynamics assessment of ß-carboline from the roots of Psammosilene tunicoides as analgesic compound.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Psammosilene tunicoides (W. C. Wu et C. Y. Wu) is a well-known medicinal herb for the treatment of pain, hemostasia and rheumatoid arthritis among Chinese people. AIM OF THE STUDY: The present study aimed to investigate the antinociceptive activity and mechanism of ß-carboline alkaloids 1-4 which were extracted from the roots of P. tunicoides. MATERIALS AND METHODS: The analgesic effects were evaluated using peripheral and central pain mouse models of nociception, including the formalin test and the tail flick test. The levels of glutamic acid (Glu) and nitric oxide (NO) in cerebellar cortexes and spinal cords (L4-6) were determined. The anti-inflammatory of all compounds were then assessed on RAW264.7 cells. RESULTS: Our results showed that compounds 1-4 had significant analgesic effects on both phases of formalin test of mice. Furthermore, all compounds showed suppressive effects on Glu in the brain and NO levels in the brain cortex and the spinal cord. Except for compound 1, the others could extend the pain threshold of hot water tail-flick in mice. In addition, compounds 2 and 3 (60 µmol/kg) could decrease GABAAα1 protein levels in spinal cord. All compounds exhibited anti-inflammatory effects by inhibiting lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells with half-maximal inhibitory concentration (IC50) 1.1-34.9 µM. CONCLUSION: ß-carboline alkaloids from the roots of P. tunicoides had significant analgesic activity by both central and peripheral mechanisms. Our findings suggested that regulating the release of NO or Glu or GABAα1 are some of the mechanisms of analgesic activity of ß-carboline alkaloids.

Study on Chinese patent medicine based on major component analysis and quality control evaluation: A case study of Jizhi Syrup.

Jizhi Syrup (JZS) is a popular Chinese patent medicine (CPM) for the treatment of respiratory diseases in clinical practice, especially acute or chronic bronchitis. JZS is a complex formula composed of 8 kinds of herbs and lack of comprehensive researches on chemical components. To further define its components, ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) and headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) were utilized to identify and classify the chemical components of JZS. A total of 178 chemical compounds encompassing the 8 herbs of JZS were identified and the chemical components were comprehensively explicit. It made up for the gap that volatile components were not studied in the previous study. Based on this, a new method for the quality control of JZS based on its characteristic components was established by fingerprints, multi-component quantitative analysis and quantity transfer of JZS. A dual-wavelength high-performance liquid chromatography (HPLC) fingerprints were established at 210 nm and 260 nm. Four volatile components (linalool, bornyl acetate, 2-undecanone and α-terpineol) and eight nonvolatile components (ephedrine hydrochloride, protocatechuic acid, 5-caffeoylquinic acid, 4-hydroxybenzoic acid, naringin, neohesperidin, glycyrrhizic acid and praeruptorin A) were quantitated by HS-SPME-GC-MS and HPLC-diode array detection (DAD). Meanwhile, six exclusive nonvolatile components were studied for the quantity transfer of Herbs-Intermediate-CPM and all the transfer rates were between 55.23% and 89.20%. This study is the first comprehensive study of the major components in JZS, and its results can be useful to standardize the quality control and provide a valuable reference for other CPMs.

Bioactive constituents from the leaves of Metapanax delavayi with anti-benign prostatic hyperplasia activities.

In the course of our continuing search for biologically active compounds from medicinal sources, we further investigated the aqueous extract of Metapanax delavayi (Franch.) J. Wen & Frodin (Araliaceae) leaves. Five undescribed terpene glycosides, liangwanosides B-F referring to two megastigmane glycosides, one monoterpene glycoside, and two sesquiterpene glycosides, together with seven known compounds were isolated. Their chemical structures were elucidated by detailed spectroscopy (1D/2D NMR), HRESIMS data analysis, hydrolysis, and comparison of experimental and calculated electronic circular dichroism (ECD) data. The biological evaluation of benign prostate hyperplasia (BPH) inhibition revealed that some compounds, including liangwanosides B-D, benzyl-O-α-L-rhamnopyranosyl-(1 â†’ 6)-ß-D-glucopyranoside, methyl 2-O-ß-D-glucopyranosylbenzoate, and 3,4-dihydroxybenzaldehyde, exhibited moderate inhibitory activity against BPH-1 cells with inhibition rates ranging from 13.8% to 23.8% at 100 µM. Among them, liangwanoside B showed the comparable effect to positive control (finasteride) at 100 µM, and its possible mechanism was then explored by molecular docking studies.

Ginsenoside Rb1 ameliorates Glycemic Disorder in Mice With High Fat Diet-Induced Obesity via Regulating Gut Microbiota and Amino Acid Metabolism.

Accumulating evidences suggested an association between gut microbiome dysbiosis and impaired glycemic control. Ginsenoside Rb1 (Rb1) is a biologically active substance of ginseng, which serves anti-diabetic effects. However, its working mechanism especially interaction with gut microbes remains elusive in detail. In this study, we investigated the impact of Rb1 oral supplementation on high fat diet (HFD) induced obesity mice, and explored its mechanism in regulating blood glucose. The results showed that higher liver weight and lower cecum weight were observed in HFD fed mice, which was maintained by Rb1 administration. In addition, Rb1 ameliorated HFD induced blood lipid abnormality and improved insulin sensitivity. Several mRNA expressions in the liver were measured by quantitative real-time PCR, of which UCP2, Nr1H4, and Fiaf were reversed by Rb1 treatment. 16S rRNA sequencing analysis indicated that Rb1 significantly altered gut microbiota composition and increased the abundance of mucin-degrading bacterium Akkermansia spp. compared to HFD mice. As suggested via functional prediction, amino acid metabolism was modulated by Rb1 supplementation. Subsequent serum amino acids investigation indicated that several diabetes associated amino acids, like branched-chain amino acids, tryptophan and alanine, were altered in company with Rb1 supplementation. Moreover, correlation analysis firstly implied that the circulation level of alanine was related to Akkermansia spp.. In summary, Rb1 supplementation improved HFD induced insulin resistance in mice, and was associated with profound changes in microbial composition and amino acid metabolism.

New tirucallane-type triterpenoids from the resin of Boswellia carteriiand their NO inhibitory activities.

Six new tirucallane-type triterpenoids (1-6), along with ten known triterpenoids, were isolated from methylene chloride extract of the resin of Boswellia carterii Birdw. By the application of the comprehensive spectroscopic data, the structures of the compounds were clarified. The experimental electronic circular dichroism spectra were compared with those calculated, which allowed to assign the absolute configurations. Compounds 5 and 6 possesed a 2, 3-seco tirucallane-type triterpenoid skeleton, which were first reported. Their inhibitory activity against NO formation in LPS-activated BV-2 cells were evaluated. Compound 9 showed appreciable inhibitory effect, with an IC50 value of 7.58 ± 0.87 µmol·L-1.

An integrated strategy of MS-network-based offline 2DLC-QTOF-MS/MS coupled with UHPLC-QTRAP®-MS/MS for the characterization and quantification of the non-polysaccharides in Sijunzi decoction.

Sijunzi decoction (SJZD), a classic recipe in traditional Chinese medicine (TCM), has been applied for the clinical treatment of gastrointestinal diseases. While there are reports on pharmaceutical substances of SJZD focusing on its polysaccharides, the composition of non-polysaccharides (NPSs) has not yet been holistically clarified. In the current study, offline two-dimensional liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (2DLC-QTOF-MS/MS) was used for comprehensive NPS chemical profiling of SJZD. In addition, the MS-network of SJZD was proposed, which led to the construction of a larger in-house chemical library and accelerated qualitative processing. Four hundred forty-nine components, among which 6 were potentially novel, and 32 were confirmed by standard substances, were identified or tentatively assigned. Furthermore, based on good method validation, 19 representative components were simultaneously quantified by ultra-high-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (UHPLC-QTRAP®-MS/MS). They were selected for quantification on the account of their bioactive reports on in vivo or in vitro activities, the peak intensity in the mass spectrum, and characteristic structures, which have the potential to be qualitative or quantitative markers of SJZD. The present work furthers understanding of the pharmacological effects and action mechanism of NPSs in SJZD, and provides a useful analytical approach for complex composition research of TCMs.

Anti-inflammatory neo-Clerodane Diterpenoids from Ajuga pantantha.

Eight new neo-clerodane diterpenoids (1-8) were acquired from the aerial parts of Ajuga pantantha. Spectroscopic data analysis permitted the definition of their structures, and experimental and calculated electronic circular dichroism data were used to define their absolute configurations. Compounds 2 and 4-8 were found to have NO inhibitory effects with IC50 values of 20.2, 45.5, 34.0, 27.0, 45.0, and 25.8 µM, respectively. The more potent compounds 2, 6, and 8 were analyzed to establish their anti-inflammatory mechanism, including regulation of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins as well as their binding interactions with the two proteins.

Withanolides from Physalis peruviana showing nitric oxide inhibitory effects and affinities with iNOS.

A phytochemical study to obtain new nitric oxide (NO) inhibitors resulted in the isolation of five new withanolides from the whole plants of Physalis peruviana. The structures were determined on the basis of extensive NMR spectroscopic data analysis as well as the time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The NO inhibitory effects were examined by inhibiting NO release in lipopolysaccharide-stimulated murine microglial BV-2 cells. Molecular docking studies showed the strong interactions of bioactive compounds with the inducible nitric oxide synthase (iNOS) protein, revealing the potential mechanism of NO inhibition of bioactive compounds.

Seco-labdane diterpenoids from the leaves of Callicarpa nudiflora showing nitric oxide inhibitory activity.

Nine previously undescribed seco-labdane diterpenoids, nudiflopenes A-I, were isolated from the leaves of Callicarpa nudiflora. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of these compounds were established by the modified Mosher's method and experimental and calculated electronic circular dichroism spectra. Nudiflopenes A-I belong to the class of seco-labdane diterpenoids. All of the isolates showed inhibitory activities on lipopolysaccharide-induced nitric oxide (NO) production in murine microglial BV-2 cells. The possible mechanism of NO inhibition of some bioactive compounds was also investigated using molecular docking, which revealed interactions of bioactive compounds with the inducible nitric oxide synthase (iNOS) protein.

NO inhibitors function as potential anti-neuroinflammatory agents for AD from the flowers of Inula japonica.

The extensive pathology studies revealed that Alzheimer's disease (AD) is closely related to neuroinflammation and anti-neuroinflammatory agents may be potentially useful for the treatment of AD. Inula japonica is a member of the Asteraceae plant family and its flowers have been used as a healthy tea and a traditional Chinese medicine. Our continuous search for new nitric oxide (NO) inhibitory substances as anti-neuroinflammatory agents for AD resulted in the isolation of two new sesquiterpenes and ten known terpenes from the flowers of I. japonica. Their structures were established on the basis of extensive analysis of NMR and MS spectroscopic data, as well as calculated and experimental electronic circular dichroism (ECD) spectra. Among these isolates, compound 1 is a new sesquiterpene with a rare tricyclic fused skeleton, and 2 processes a 1,10-seco-eudesmane skeleton. The anti-neuroinflammatory effects were examined by inhibiting NO release in LPS-induced murine microglial BV-2 cells. The possible mechanism of NO inhibition was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the iNOS protein. The present study disclosed that the flowers of I. japonica as a healthy tea are potentially useful for AD and related neuroinflammatory diseases.

Daphnane diterpenoids with nitric oxide inhibitory activities and interactions with iNOS from the leaves of Trigonostemon thyrsoideus.

A phytochemical investigation to search for new nitric oxide (NO) inhibitors resulted in the isolation of seven previously undescribed daphnane diterpenoids, thyrsoidpenes A-G, from the leaves of Trigonostemon thyrsoideus. Their structures including absolute configurations were elucidated on the basis of extensive NMR spectroscopic data analysis and the time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. Thyrsoidpenes B-G feature rare polycyclic caged structures of daphnane diterpenoid orthoester. The NO inhibitory effects were examined and all of the compounds showed inhibitory activities toward LPS-induced NO production in murine microglial BV-2 cells. The possible mechanism of NO inhibition of some bioactive compounds was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the iNOS protein.

NO inhibitory constituents as potential anti-neuroinflammatory agents for AD from Blumea balsamifera.

Our continuous search for new nitric oxide (NO) inhibitory substances as anti-neuroinflammatory agents for AD resulted in the isolation of one new labdane diterpenoid and three new guaiane sesquiterpenoids, as well as ten known compounds from Blumea balsamifera. Their structures were elucidated by NMR spectroscopic data analysis and the time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The anti-neuroinflammatory effects were examined by inhibiting NO release in LPS-induced murine microglial BV-2 cells. The possible mechanism of NO inhibition of some bioactive compounds was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the iNOS protein.

Phytochemicals with NO inhibitory effects and interactions with iNOS protein from Trigonostemon howii.

A phytochemical investigation to obtain new NO inhibitors led to the isolation of nine compounds including one new guaiane-type sesquiterpenoid (1) and two new cleistanthane diterpenoids (2 and 3) from the stems of Trigonostemon howii. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of new compounds 1-3 were established via comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 2 and 3 possess a rare 3,4-seco-cleistanthane diterpenoid skeleton. All of the compounds showed inhibitory effects on lipopolysaccharide-induced NO production in murine microglial BV-2 cells. The further molecular docking studies indicated the strong interactions between some bioactive compounds with the iNOS protein, which revealed the possible and potential mechanism of NO inhibition of bioactive compounds.